HK1222678B - Cd19 specific chimeric antigen receptor and uses thereof - Google Patents

Description

CD19-specific chimeric antigen receptor and its use

Field of the Invention

The present invention relates to chimeric antigen receptors (CARs). CARs are able to utilize the properties of the ligand binding domain to redirect the specificity and reactivity of immune cells to selected targets. In particular, the present invention relates to chimeric antigen receptors in which the extracellular ligand binds to scFV derived from a CD19 monoclonal antibody (preferably 4G7). The present invention also relates to polynucleotides encoding the CAR, vectors, and isolated cells expressing the CAR on their surface. The present invention also relates to methods for modifying immune cells expressing 4G7-CAR on their surface to confer a prolonged "activated" state on the transduced cells. The present invention is particularly useful for treating B-cell lymphomas and leukemias.

Background of the Invention

Adoptive immunotherapy, which involves the transfer of autologous antigen-specific T cells generated in vitro, is a promising approach for treating viral infections and cancer. T cells used for adoptive immunotherapy can be generated by expanding antigen-specific T cells or reintroducing T cells through genetic modification (Park, Rosenberg et al. 2011). The transfer of viral antigen-specific T cells is a well-established procedure for treating transplant-associated viral infections and malignancies associated with rare viruses. Similarly, the isolation and transfer of tumor-specific T cells has been shown to successfully treat melanoma.

Novel specificities in T cells have been successfully obtained through gene transfer of transgenic T cell receptors or chimeric antigen receptors (CARs) (Jena, Dotti et al. 2010). CARs are synthetic receptors that consist of a targeting moiety associated with one or more signaling domains in a single fusion molecule. Generally, the binding portion of a CAR consists of the antigen binding domain of a single-chain antibody (scFv), which comprises a light and variable fragment of a monoclonal antibody joined by a flexible linker. Binding moieties based on receptor or ligand domains have also been successfully used. The signaling domain of first-generation CARs was derived from the cytoplasmic region of CD3ζ or the Fc receptor gamma chain. First-generation CARs have been shown to successfully induce T cell cytotoxicity, however, they have not been able to provide prolonged expansion and anti-tumor activity in vivo. Signaling domains from costimulatory molecules including CD28, OX-40 (CD134), and 4-1BB (CD137) are added alone (second generation) or in combination (third generation) to improve the survival rate of CAR-modified T cells and increase the proliferation of CAR-modified T cells. CARs have successfully allowed T cells to be redirected to antigens expressed on the surface of tumor cells from a variety of malignancies including lymphomas and solid tumors (Jena, Dotti et al. 2010).

CD19 is an attractive target for immunotherapy because it is expressed consistently by most B-acute lymphoblastic leukemias (B-ALL) and is absent on non-hematopoietic cells, bone marrow, erythrocytes, T cells, and bone marrow stem cells. Clinical trials targeting CD19 for B-cell malignancies have yielded encouraging anti-tumor responses. Most T cells engineered to express a chimeric antigen receptor (CAR) have been fused with the specificity of the scFv region derived from the CD19-specific mouse monoclonal antibody FMC63 (Nicholson, Lenton et al. 1997; Cooper, Topp et al. 2003; Cooper, Jena et al. 2012) (International Application: WO2013/126712). However, there remains a need for improved CAR construction that exhibits greater compatibility with T-cell proliferation to enable cells expressing such CARs to achieve significant clinical advantages.

SUMMARY OF THE INVENTION

The inventors have generated a CD19-specific CAR (4G7-CAR) comprising a scFV derived from a CD19-specific monoclonal antibody (4G7) and surprisingly discovered that introducing the resulting 4G7-CAR into primary T cells can confer a prolonged "activated" state on the transduced cells, independent of antigen binding. Following nonspecific activation in vitro (e.g., using anti-CD3/CD28-coated beads and recombinant IL2), the cells exhibited increased cell size (blast formation) and expression of activation markers (CD25) for an extended period of time compared to cells transduced with a similar CAR comprising the FMC63 scFV. This prolonged activation allows for prolonged proliferation and provides an antigen-independent mechanism for expanding 4G7-CAR cells in vitro.

The present invention therefore provides a chimeric antigen receptor comprising at least one extracellular ligand binding domain, a transmembrane domain and at least one signal transduction domain, wherein the extracellular ligand binding domain comprises an scFV derived from the specific monoclonal antibody 4G7. Specifically, the CAR of the present invention, once transduced into an immune cell, causes the activation and proliferation of the cell that is independent of the antigen. The present invention also relates to nucleic acids, vectors and methods for modifying immune cells encoding a CAR comprising an scFV derived from the CD19-specific monoclonal antibody 4G7, the method comprising introducing the 4G7 CAR into a cell. The present invention also relates to genetically modified immune cells expressing 4G7 on their surface, particularly immune cells that proliferate independently of an antigen mechanism. The genetically modified immune cells of the present invention are particularly suitable for therapeutic applications such as the treatment of B-cell lymphoma or leukemia.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Proliferation of TCRα non-activated T cells (KO) transduced with the 4G7-CAR lentiviral vector compared to non-transduced KO T cells (NTD). Proliferation was followed over 30 days after a reactivation step with soluble anti-CD28 (IL2+CD28) or without reactivation (IL2).

Figure 2: Analysis of CD25 activation marker expression on the surface of non-activated TCRα T cells transduced with 4G7-CAR lentiviral vectors, gated based on 4G7-CAR expression (CAR+, CAR-) and compared to TCRα-positive cells that were not electroporated (NEP) or that were TCRα-destroyed but not transduced (NTD). CD25 expression was analyzed after a reactivation step (IL2+CD28) or without reactivation (IL2) using soluble anti-CD28.

Figure 3: Analysis of CAR expression on the surface of T cells transduced with lentiviral vectors encoding either 4G7-CAR or FMC63-CAR. The analysis was performed by flow cytometry on days 3, 8, and 15 after transduction. NT refers to non-transduced T cells.

Figure 4: Analysis of CD25 expression on the surface of T cells transduced with lentiviral vectors encoding either 4G7-CAR or FMC63-CAR. The analysis was performed by flow cytometry on days 3, 8, and 15 after transduction. NT refers to non-transduced T cells.

Figure 5: Size analysis of T cells transduced with lentiviral vectors encoding either 4G7-CAR or FMC63-CAR. The analysis was performed by flow cytometry on days 3, 8, and 15 after transduction. NT refers to non-transduced T cells.

Figure 6: Proliferation of T cells transduced with 4G7-CAR compared to FMC63 lentiviral vectors. Proliferation was tracked over 20 days after a reactivation step with soluble anti-CD28 (CD28) or without reactivation (-). NTD refers to non-transduced T cells.

Detailed Description of the Invention

Unless specifically defined herein, all technical and scientific terms used have the same meanings as commonly understood by one skilled in the art of gene therapy, biochemistry, genetics, and molecular biology.

All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention with the appropriate methods and materials described herein. All publications, patent applications, patents, and other references described herein are incorporated by reference in their entirety. In the event of a conflict, the present specification (including definitions) will control. In addition, unless otherwise indicated, the materials, methods, and examples are illustrative only and are not intended to be limiting.

Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are fully described in the literature. See, e.g., Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, 3rd ed. (Sambrook et al., 2001, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Mullis et al., U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Harries and S. J. Higgins, eds., 1984); Transcription And Translation (B. D. Hames and S. J. Higgins, eds., 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds., Academic Press, Inc., New York), specifically, Volumes 154 and 155 (Wu et al., eds.) and Volume 185, "Gene Expression Technology" (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos, eds., Cold Spring Harbor Laboratory, 1987); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986).

CD19-specific chimeric antigen receptor

The present invention relates to a chimeric antigen receptor (CAR), which comprises an extracellular ligand-binding domain, a transmembrane domain, and a signal transduction domain.

As used herein, the term "extracellular ligand-binding domain" is defined as an oligopeptide or polypeptide capable of binding to a ligand. Preferably, the domain will be capable of interacting with a cell surface molecule. For example, the extracellular ligand-binding domain can be selected to recognize a ligand that serves as a cell surface marker on a target cell associated with a particular disease state.

In a preferred embodiment, the extracellular ligand-binding domain comprises a single-chain antibody fragment (scFv) comprising the light ( _VL ) and heavy ( _VH ) variable fragments of a target antigen-specific monoclonal antibody connected by a flexible linker. In a preferred embodiment, the scFV is derived from the CD19 monoclonal antibody 4G7 (Peipp, Saul et al., 2004). Preferably, the scFV of the present invention comprises a portion of the immunoglobulin γ1 heavy chain of the CD19 monoclonal antibody 4G7 (GenBank: CAD88275.1; SEQ ID NO: 1) and a portion of the immunoglobulin κ light chain of the CD19 monoclonal antibody 4G7 (GenBank: CAD88204.1; SEQ ID NO: 2), preferably connected by a flexible linker. In a preferred embodiment, the scFV of the present invention comprises the variable fragment of the immunoglobulin gamma 1 heavy chain of the CD19 monoclonal antibody 4G7 (SEQ ID NO: 3) and the variable fragment of the immunoglobulin kappa light chain of the CD19 monoclonal antibody 4G7 (SEQ ID NO: 4 or SEQ ID NO: 5), connected by a flexible linker. In a specific embodiment, the flexible linker comprises the amino acid sequence (SEQ ID NO: 6).

In other words, the CAR comprises an extracellular ligand-binding domain comprising a single-chain Fv fragment derived from the CD19-specific monoclonal antibody 4G7. In a specific embodiment, the scFv comprises a portion of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5. In a preferred embodiment, the scFv comprises an amino acid sequence having at least 70%, preferably at least 80%, and more preferably at least 90%, 95%, 97%, or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 8.

The signal transduction domain or intracellular signal transduction domain of the CAR according to the present invention is the reason for the intracellular signal transduction after the activation of immune cells and the immune response caused by the binding of the extracellular ligand binding domain to the target. In other words, the signal transduction domain is the reason for the activation of at least one normal effector function of the immune cells expressing CAR. For example, the effector function of T cells can be cytolytic activity or auxiliary activity (including the secretion of cytokines). Therefore, the term "signal transduction domain" refers to the transduction effector signal function signal of the protein and the part that directs the cell to perform specialized functions.

Preferred examples of signal transduction domains used in CARs can be T cell receptors and cytoplasmic sequences of co-receptors that coordinate after antigen receptor engagement to initiate signal transduction, and any derivatives or variants of the sequences with the same functional capabilities and any synthetic sequences. The signal transduction domain includes two different types of cytoplasmic signaling sequences, those that initiate the primary activation sequence independent of the antigen, and those that can act independently of the antigen to provide a secondary or costimulatory signal. The primary cytoplasmic signaling sequence may include a signaling motif of an immunoreceptor tyrosine-based activation motif called ITAM. ITAM is a well-defined signaling motif found in the cytoplasmic tail of various receptors that act as binding sites for syk/zap70 class tyrosine kinases. Examples of ITAMs used in the present invention may include, but are not limited to, those derived from TCRζ, FcRγ, FcRβ, FcRε, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. In a preferred embodiment, the signal transduction domain of the CAR may include a CD3ζ signaling domain having an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% sequence identity relative to an amino acid sequence selected from the group consisting of (SEQ ID NO: 10).

In a specific embodiment, the signal transduction domain of the CAR of the present invention includes a co-stimulatory signal molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands required for an effective immune response. "Co-stimulatory ligand" refers to a molecule on an antigen presenting cell that specifically binds to a cognate co-stimulatory molecule on a T-cell, thereby providing a signal other than the primary signal provided by, for example, binding of the TCR/CD3 complex to a peptide-loaded MHC molecule, which mediates T cell responses including (but not limited to) proliferation activation, differentiation, etc. Co-stimulatory ligands may include (but are not limited to) CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecules (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, and Toll ligand receptor. Co-stimulatory ligands also include antibodies that specifically bind to co-stimulatory molecules on T cells, such as, but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

"Costimulatory molecules" refer to cognate binding partners that specifically bind to costimulatory ligands on T-cells, thereby mediating costimulatory responses such as, but not limited to, proliferation by the cells. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors. Examples of costimulatory molecules include CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, antigen-1 associated with lymphocyte function (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

In a preferred embodiment, the signal transduction domain of the CAR of the present invention comprises a portion of a costimulatory signal molecule selected from the group consisting of fragments of 4-1BB (GenBank: AAA53133) and CD28 (NP_006130.1). Specifically, the signal transduction domain of the CAR of the present invention comprises an amino acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 12.

According to the present invention, CAR is expressed on the surface membrane of the cell. Therefore, CAR may include a transmembrane domain. The distinguishing features of a suitable transmembrane domain include expression on the surface of cells, preferably immune cells in the present invention, specifically lymphocytes or natural killer (NK) cells, and interacting together to direct the ability of immune cells to respond to the cells of predetermined target cells. The transmembrane domain can be derived from natural or synthetic sources. The transmembrane domain can be derived from any membrane-bound or transmembrane protein. As a non-limiting example, a transmembrane polypeptide can be a subunit of a T cell receptor (such as α, β, γ or δ), a subunit chain constituting a CD3 complex, IL2 receptor p55 (α chain), p75 (β chain) or γ chain, Fc receptor (specifically Fcγ receptor III) or a polypeptide of a CD protein. Alternatively, the transmembrane domain can be synthetic and can mainly include hydrophobic residues, such as leucine and valine. In a preferred embodiment, the transmembrane domain is derived from human CD8α chain (e.g., NP_001139345.1). The membrane-spanning domain may further include a stem region between the extracellular ligand-binding domain and the membrane-spanning domain. The term "stem region" as used herein generally refers to any oligopeptide or polypeptide that functions to connect a membrane-spanning domain to an extracellular ligand-binding domain. Specifically, the stem region is used to provide more flexibility and accessibility for the extracellular ligand-binding domain. The stem region may include up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. The stem region may be derived from all or part of a naturally occurring molecule, such as, all or part of the extracellular region derived from CD8, CD4 or CD28, or all or part of an antibody constant region. Alternatively, the stem region may be a synthetic sequence equivalent to a naturally occurring stem sequence, or may be a whole synthetic stem sequence. In a preferred embodiment, the stem region is a portion of a human CD8 α chain (e.g., NP_001139345.1) (SEQ ID NO: 196). In another specific embodiment, the transmembrane and hinge domains comprise a portion of the human CD8 α chain, preferably comprising at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 13.

In a specific embodiment, the chimeric antigen receptor of the present invention comprises a scFV derived from the CD19 monoclonal antibody 4G7, a CD8α human hinge and transmembrane domain, a CD3ζ signaling domain, and a 4-1BB signaling domain. Preferably, the 4G7 CAR of the present invention comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 15 having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99% sequence identity.

Downregulation or mutation of the target antigen is often observed in cancer cells, resulting in antigen loss escape variants. Therefore, to offset tumor escape and make immune cells more specific to the target, the CD19-specific CAR may include another extracellular ligand binding domain to simultaneously bind to different elements in the target, thereby enhancing immune cell activation and function. In one embodiment, the extracellular ligand binding domain can be placed in series on the same transmembrane polypeptide and optionally separated by a linker. In another embodiment, the different extracellular ligand binding domains can be placed on different transmembrane polypeptides constituting the CAR. In another embodiment, the present invention relates to a CAR group, each of which comprises different extracellular ligand binding domains. In a specific embodiment, the present invention relates to a method for modifying immune cells, the method comprising providing an immune cell and expressing a CAR group at the surface of the cell, each of which comprises different extracellular ligand binding domains. In another specific embodiment, the present invention relates to a method for modifying immune cells, the method comprising providing an immune cell and introducing a polynucleotide encoding a polypeptide into the cell, the polypeptide constituting a CAR group each comprising different extracellular ligand binding domains. The term "CAR population" refers to at least two, three, four, five, six, or more CARs, each of which comprises a different extracellular ligand-binding domain. The different extracellular ligand-binding domains according to the present invention can preferably bind to different elements in the target simultaneously, thereby enhancing immune cell activation and function. The present invention also relates to isolated immune cells comprising a CAR population, each of which comprises a different extracellular ligand-binding domain.

Polynucleotides, vectors:

The present invention also relates to polynucleotides and vectors encoding the CAR according to the present invention as described above. In a preferred embodiment, the present invention relates to a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 17. In a preferred embodiment, the polynucleotide has at least 70%, preferably at least 80%, and more preferably at least 90%, 95%, 97%, or 99% sequence identity with a nucleic acid sequence selected from the group consisting of SEQ ID NO: 17.

The polynucleotide can be an expression cassette or expression vector (e.g., a plasmid for introduction into a bacterial host cell, or a viral vector such as a baculovirus vector for transfection of an insect host cell, or a plasmid or viral vector such as a lentivirus for transfection of a mammalian host cell).

In certain embodiments, the different nucleic acid sequences may be contained within a single polynucleotide or vector that includes a nucleic acid sequence encoding a ribosomal skip sequence (such as a sequence encoding a 2A peptide). The 2A peptide, identified in the foot-and-mouth disease virus subgroup of the picornavirus, allows the ribosome to "skip" from one codon to the next without forming a peptide bond between the two amino acids encoded by the codon (see (Donnelly and Elliott 2001; Atkins, Wills et al. 2007; Doronina, Wu et al. 2008)). A "codon" is defined as the three nucleotides in an mRNA (or on the sense strand of a DNA molecule) that are translated by the ribosome into one amino acid residue. Thus, when the polypeptides are separated by an in-frame 2A oligopeptide sequence, two polypeptides can be synthesized from a single, continuous open reading frame in an mRNA. This ribosomal skipping mechanism is well known in the art and is used by several vectors to express proteins encoded by a single messenger RNA.

To direct a transmembrane polypeptide into the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence, prepro sequence, or presequence) is provided in a polynucleotide sequence or vector sequence. The secretory signal sequence is operably linked to the transmembrane nucleic acid sequence, that is, the two sequences are combined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. The secretory signal sequence is typically located 5' of the nucleic acid sequence encoding the polypeptide of interest, although certain secretory signal sequences may be located at other positions in the nucleic acid sequence of interest (see, e.g., Welch et al., U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830). In a preferred embodiment, the signal peptide comprises the amino acid sequence SEQ ID NO: 18 and 19.

Those skilled in the art will recognize that due to the degeneracy of the genetic code, considerable sequence variation is possible in these polynucleotide molecules. Preferably, the nucleic acid sequences of the present invention are codon-optimized for expression in mammalian cells, preferably human cells. Codon optimization refers to the exchange of codons in the sequence of interest that are typically rare in highly expressed genes of a given species with codons that are typically common in highly expressed genes of that species. When the codons are exchanged, the codons encode amino acids.

In a preferred embodiment, the polynucleotide according to the present invention comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 17. The present invention relates to a polynucleotide comprising a nucleic acid sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% sequence identity with a nucleic acid sequence selected from the group consisting of SEQ ID NO: 17.

Methods for modifying immune cells:

In a specific embodiment, the present invention relates to a method for producing an immune cell for immunotherapy, comprising introducing a CAR according to the present invention into the immune cell and expanding the cell. In a specific embodiment, the present invention relates to a method for modifying an immune cell, comprising providing a cell and expressing at least one CAR as described above on the surface of the cell. In a specific embodiment, the method comprises transforming the cell with at least one polynucleotide encoding a CAR as described above and expressing the polynucleotide into the cell.

In a preferred embodiment, the polynucleotide is contained in a lentiviral vector for stable expression in cells.

In another embodiment, the method further comprises the step of genetically modifying the cell by inactivating at least one gene expressing a component of the TCR, a target of an immunosuppressant, an HLA gene, and/or an immune checkpoint gene such as PDCD1 or CTLA-4. In a preferred embodiment, the gene is selected from the group consisting of TCRα, TCRβ, CD52, GR, PD1, and CTLA-4. In a preferred embodiment, the method further comprises introducing into the T cell a rare-cutting nuclease that can selectively inactivate the gene by DNA cleavage. In a more preferred embodiment, the rare-cutting nuclease is a TALE-nuclease or a Cas9 nuclease.

Delivery Method

The above-mentioned different methods involve introducing CAR into cells. As a non-limiting example, the CAR can be introduced as a transgene encoded by a plasmid vector. The plasmid vector can also contain a selection marker that provides recognition and/or selection of cells that receive the vector.

The polypeptide can be synthesized in situ in the cell by introducing a polynucleotide encoding the polypeptide into the cell. Alternatively, the polypeptide can be produced extracellularly and then introduced therein. Methods for introducing polynucleotide constructs into cells are known in the art and include, as non-limiting examples, stable transformation methods in which the polynucleotide construct is integrated into the genome of the cell, transient transformation methods in which the polynucleotide construct is not integrated into the genome of the cell, and virus-mediated methods. The polynucleotide can be introduced into the cell by, for example, a recombinant viral vector (e.g., retrovirus, adenovirus), liposomes, etc. For example, transient transformation methods include, for example, microinjection, electroporation, or particle bombardment. In view of being expressed in the cell, the polynucleotide can be contained in a vector, more specifically a plasmid or a virus.

Engineered immune cells

The present invention also relates to isolated cells or cell lines that can be easily obtained by this method to transform cells. Specifically, the isolated cells contain at least one of the above-mentioned CARs. In another embodiment, the isolated cells contain a population of CARs each containing a different extracellular ligand binding domain. Specifically, the isolated cells contain an exogenous polynucleotide sequence encoding a CAR. The genetically modified immune cells of the present invention are activated and proliferated independently of the antigen binding mechanism.

Also included within the scope of the present invention are isolated immune cells, preferably T cells obtained according to any of the above methods. Such immune cells refer to hematopoietic cells that are functionally involved in the initiation and/or execution of innate and/or adaptive immune responses. The immune cells according to the present invention may be derived from stem cells. These stem cells may be adult stem cells, non-human embryonic stem cells, more specifically non-human stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells. Representative human cells are CD34+ cells. The isolated cells may also be dendritic cells, killer dendritic cells, mast cells, NK cells, B cells, or T cells selected from the group consisting of inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, or helper T lymphocytes. In another embodiment, the cells may be derived from the group consisting of CD4+ T lymphocytes and CD8+ T lymphocytes. Prior to expansion and genetic modification of the cells of the present invention, the cells may be obtained from a subject via a variety of non-limiting methods. Cells can be obtained from a variety of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the injection site, ascites, pleural effusion, spleen tissue and tumors. In certain embodiments of the present invention, any number of T cell lines available and known to those skilled in the art can be used. In another embodiment, the cells can be derived from a healthy donor, a patient diagnosed with cancer, or a patient diagnosed with an infection. In another embodiment, the cells are part of a mixed cell population exhibiting different phenotypic characteristics. Also included within the scope of the present invention are cell lines obtained from transformed T-cells according to the above-described methods. Modified cells that are resistant to immunosuppressive therapy and readily obtained by the above-described methods are included within the scope of the present invention.

In another embodiment, the isolated cell according to the present invention comprises a polynucleotide encoding a CAR.

T cell activation and expansion

Whether before or after genetic modification of T cells, even if the genetically modified immune cells of the present invention are activated and proliferated independently of an antigen binding mechanism, immune cells, specifically T cells of the present invention, can be further generally activated and expanded using methods such as those described in, for example, U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005. T cells can be expanded in vitro or in vivo.

Generally, T cells of the present invention are expanded by contacting an agent that stimulates the CD3 TCR complex and a co-stimulatory molecule on the surface of the T cell to generate an activation signal for the T cell.

For example, chemicals such as the calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogenic lectins such as phytohemagglutinin (PHA) can be used to generate activation signals for T-cells.

As a non-limiting example, T cell populations can be stimulated in vitro, such as by contact with anti-CD3 antibodies, or antigen-binding fragments thereof, or anti-CD2 antibodies immobilized on a surface, or by contact with protein kinase C activators (e.g., bryostatin) together with calcium ionophores. For costimulation of accessory molecules on the surface of T cells, ligands that bind to accessory molecules are used. For example, T cell populations can be contacted with anti-CD3 antibodies and anti-CD28 antibodies under conditions suitable for stimulating the proliferation of T cells. Suitable conditions for T cell culture include suitable culture media (e.g., minimal essential medium or RPMI medium 1640 or X-vivo 5 (Lonza)) that can contain factors required for proliferation and survival, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-g, IL-4, IL-7, GM-CSF, -10, -2, IL-15, TGF-β, and TNF- or any other additives for cell growth known to those skilled in the art. Other additives for cell growth include, but are not limited to, surfactants, human plasma protein powder (plasmanate), and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. Culture media can include serum-free or supplemented with appropriate amounts of serum (or plasma) or a defined set of hormones, and/or cytokines sufficient to grow and expand T cells, with added amino acids, sodium pyruvate, and vitamins, such as RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1, and X-Vivo 20 (Optimizer). Antibiotics such as penicillin and streptomycin are included only in experimental culture media, not in cultures of cells intended for injection into subjects. Target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% _CO2 ). T cells exposed multiple times to different stimuli can exhibit different characteristics.

In another specific embodiment, the cells can be expanded by co-culturing with tissues or cells. The cells can also be expanded in vivo, for example, in the blood of a subject after the cells are administered to the subject.

Therapeutic applications

In another embodiment, the isolated cells obtained by various methods or cell lines derived from the isolated cells described above can be used as medicaments. In another embodiment, the medicaments can be used to treat cancer in patients in need thereof, particularly B-cell lymphomas and leukemias in patients in need thereof. In another embodiment, the isolated cells or cell lines derived from the isolated cells according to the present invention can be used to manufacture medicaments for treating cancer in patients in need thereof.

In another aspect, the present invention is based on a method for treating a patient in need thereof, the method comprising at least one of the following steps:

(a) providing immune cells obtainable by any one of the above methods;

(b) administering the transformed immune cells to the patient,

In one embodiment, the T cells of the present invention can undergo robust in vivo T cell expansion and can be sustained for an extended period of time.

The treatment may be ameliorative, curative, or preventative. It may be part of an autologous immunotherapy or part of an allogeneic immunotherapy. By autologous, it is meant that the cells, cell lines, or cell populations used to treat the patient are derived from the patient or from a donor who is human leukocyte antigen (HLA) compatible. By allogeneic, it is meant that the cells or cell populations used to treat the patient are not derived from the patient but from a donor.

Cells that can be used with the disclosed methods are described in the previous sections. The treatment can be used to treat patients diagnosed with cancer. Treatable cancers may include non-solid tumors (such as hematological tumors, including but not limited to pre-B ALL (pediatric indications), adult ALL, mantle cell lymphoma, diffuse large B-cell lymphoma, etc. The types of cancer that can be treated by the CAR of the present invention include but are not limited to certain leukemias or lymphoid malignancies. It also includes adult tumors/cancers and childhood tumors/cancers.

The treatment may be a treatment in combination with one or more anti-cancer therapies selected from the group consisting of antibody therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy, laser therapy, and radiation therapy.

According to a preferred embodiment of the present invention, the treatment can be administered to patients undergoing immunosuppressive therapy. In practice, the present invention preferably relies on cells or cell populations that have become resistant to at least one immunosuppressant by inactivating a gene encoding a receptor for the immunosuppressant. In this regard, immunosuppressive therapy should facilitate the selection and expansion of T cells according to the present invention in patients.

The cells or cell populations according to the present invention can be administered by any convenient method, including by aerosol inhalation, injection, ingestion, infusion, implantation, or transplantation. The compositions described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intramedullaryly, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally. In one embodiment, the cell compositions of the present invention are preferably administered by intravenous injection.

The administration of cells or cell groups can consist of administering 10 ⁴ to 10 ⁹ cells/kg body weight, preferably 10 ⁵ to 10 ⁶ cells/kg body weight, including all integer values of the number of cells in the range. The cells or cell groups can be administered in one or more doses. In another embodiment, the effective amount of cells is administered in a single dose. In another embodiment, the effective amount of cells is administered in more than one dose over a period of time. The timing of administration is at the discretion of the attending physician and depends on the patient's clinical condition. Cells or cell groups can be obtained from any source such as a blood bank or a donor. Although individual needs vary, the determination of the optimal range of effective amounts for a given cell type for a specific disease or condition is within the art. An effective amount means an amount that provides a therapeutic or preventive benefit. The dosage administered will depend on the recipient's age, health status, and weight, the type of concurrent treatment (if any), the frequency of treatment, and the nature of the desired effect.

In another embodiment, the effective amount of cells or a composition comprising those cells is administered parenterally. The administration can be intravenous. The administration can be performed directly by injection into the tumor.

In certain embodiments of the invention, the cells are administered to a patient in conjunction with (e.g., prior to, concurrently with, or after) any number of related therapeutic modalities, including, but not limited to, treatment with active agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C), or natalizimab for MS patients, or efaliztimab for psoriasis patients, or other treatments for PML patients. In other embodiments, the T cells of the invention can be administered in conjunction with chemotherapy, radiation, immunosuppressants (such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506), antibodies or other immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytotoxins, fludaribine, cyclosporine, FK506, rapamycin, mycophenolate mofetil, or other anti-inflammatory drugs. Acid), steroids, FR901228, cytokines, and radiation are used in combination. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase (rapamycin), which is important for growth factor-induced signaling (Henderson, Naya et al. 1991; Liu, Albers et al. 1992; Bierer, Hollander et al. 1993). In other embodiments, the cell compositions of the present invention are used in conjunction with (e.g., before, simultaneously with, or after) bone marrow transplantation, the use of chemotherapeutic agents such as fludarabine, or the administration of cytokines. In some embodiments, T cell ablative therapy, including abine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies (such as OKT3 or CAMPATH), is administered to the patient. In another embodiment, the cell compositions of the present invention are administered after B cell ablative therapy, such as an agent reactive with CD20 (e.g., Rituxan). For example, in one embodiment, the subject may undergo standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant. In certain embodiments, after the transplant, the subject receives an infusion of the expanded immune cells of the present invention. In another embodiment, the expanded cells are administered before or after surgery.

Other definitions

Unless otherwise indicated, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one. Amino acid residues in polypeptide sequences are designated herein according to a one-letter code in which, for example, Q means a Gln or glutamine residue, R means an Arg or arginine residue, and D means an Asp or aspartic acid residue.

Amino acid substitution means that one amino acid residue is replaced by another amino acid residue, for example, an arginine residue in a peptide sequence is replaced by a glutamine residue.

Nucleotides are named as follows: A one-letter code is used to name the nucleoside bases: a for adenine, t for thymine, c for cytosine, and g for guanine. For degenerate nucleotides, r represents g or a (purine nucleotides), k represents g or t, s represents g or c, w represents a or t, m represents a or c, y represents t or c (pyrimidine nucleotides), d represents g, a or t, v represents g, a or c, b represents g, t or c, h represents a, t or c, and n represents g, a, t or c.

As used herein, "nucleic acid" or "polynucleotide" refers to nucleotides and/or polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by polymerase chain reaction (PCR), and fragments generated by any of ligation, cleavage, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally occurring nucleotides (such as DNA and RNA), or analogs of naturally occurring nucleotides (e.g., enantiomeric forms of naturally occurring nucleotides), or a combination of both. Modified nucleotides can have changes in the sugar moiety and/or in the pyrimidine or purine base moiety. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyls, amines, and azides, or functionalization of sugars as ethers or esters. In addition, the entire sugar moiety can be replaced by sterically and electrically similar structures (such as azasugars and carbocyclic sugar analogs). Examples of modifications in the base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substituents. Nucleic acid monomers can be linked via phosphodiester bonds or analogs of such linkages. Nucleic acids can be single-stranded or double-stranded.

-The so-called chimeric antigen receptor (CAR) is a desired molecule that combines a binding domain for a component present on a target cell (e.g., an antibody-based specificity for a desired antigen (e.g., a tumor antigen)) with a T cell receptor activating intracellular domain to generate a chimeric protein that exhibits specific anti-target cell immune activity. In general, CAR consists of an extracellular single-chain antibody (scFvFc) fused to the intracellular signaling domain of the ζ chain (scFvFc:ζ) of the T cell antigen receptor complex and has the ability to redirect antigen recognition based on the specificity of a monoclonal antibody when expressed in T cells. An example of a CAR used in the present invention is a CAR directed against the CD19 antigen and can, as a non-limiting example, comprise the amino acid sequence: SEQ ID NO: 14.

-The term "endonuclease" refers to any wild-type or variant enzyme that can catalyze the hydrolysis (cleavage) of bonds between DNA or RNA molecules, preferably nucleic acids in DNA molecules. Endonucleases do not cut DNA or RNA molecules regardless of their sequence, but can recognize and cut DNA or RNA molecules at specific polynucleotide sequences, also known as "target sequences" or "target sites." Endonucleases can be classified as rare-cutting endonucleases when they typically have a polynucleotide recognition site that is more than 12 base pairs (bp) in length, more preferably 14 to 55 bp in length. Rare-cutting endonucleases significantly increase HR by inducing DNA double-strand breaks (DSBs) at defined loci (Perrin, Buckle et al. 1993; Rouet, Smih et al. 1994; Choulika, Perrin et al. 1995; Pingoud and Silva 2007). Rare-cutting endonucleases can be, for example, homing endonucleases (Paques and Duchateau 2007), chimeric zinc finger nucleases (ZFNs) derived from the fusion of engineered zinc finger domains to the catalytic domain of a restriction enzyme such as Fok1 (Porteusy and Carrot 2005), Cas9 endonucleases from the CRISPR system (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012; Cong, Ran et al. 2013; Mali, Yang et al. 2013), or chemical endonucleases (Eisenschmidt, Lanio et al. 2005; Arimondo, Thomas et al. 2006). In chemical endonucleases, a chemical or peptide cleavage agent is conjugated to a polymer of nucleic acid or to another DNA that recognizes a specific target sequence, thereby targeting the cleavage activity to the specific sequence. Chemical endonucleases also include synthetic nucleases, for example, o-phenanthroline, DNA cleavage molecules, and conjugates of triple helix-forming oligonucleotides (TFOs) known to bind to specific DNA sequences (Kalish and Glazer 2005). Such chemical endonucleases are encompassed by the term "endonuclease" according to the present invention.

- "TALE-nuclease" (TALEN) means a fusion protein consisting of a nucleic acid binding domain, typically derived from a transcription activator-like effector (TALE), and a nuclease catalytic domain that cleaves a nucleic acid target sequence. The catalytic domain is preferably a nuclease domain and more preferably a domain with endonuclease activity, such as I-Tevl, ColE7, NucA and Fok-I. In a specific embodiment, the TALE domain may be fused to a large range of nucleases such as, for example, I-Crel and I-Onul or functional variants thereof. In a more preferred embodiment, the nuclease is a monomeric TALE-nuclease. Monomeric TALE-nucleases are TALE-nucleases that do not require dimerization for specific recognition and cleavage, such as, for example, a fusion of the engineered TAL repeats described in WO2012138927 with the catalytic domain of I-Tevl. Transcription activator-like effectors (TALEs) are proteins from the bacterial species Xanthomonas that contain multiple repeats, each containing a diresidue (RVD) at positions 12 and 13 that is specific for a respective nucleotide base of the sequence being targeted by the nucleic acid. Binding domains (MBBBDs) with similar modular base-base nucleic acid binding properties can also be derived from novel modular proteins recently discovered by the applicants in different bacterial species. These novel modular proteins have the advantage of exhibiting greater sequence variability than TAL repeats. Preferably, the RVDs associated with recognition of different nucleotides are HD for recognizing C, NG for recognizing T, NI for recognizing A, NN for recognizing G or A, NS for recognizing A, C, G or T, HG for recognizing T, IG for recognizing T, NK for recognizing G, HA for recognizing C, ND for recognizing C, HI for recognizing C, HN for recognizing G, NA for recognizing G, SN for recognizing G or A or YG for recognizing T, TL for recognizing A, VT for recognizing A or G, and SW for recognizing A. In another embodiment, key amino acids 12 and 13 can be mutated to other amino acid residues to adjust their specificity for the nucleotides A, T, C and G, in other words, to improve the specificity. TALE-nucleases have been described and used to facilitate gene targeting and gene modification (Both, Scholze et al. 2009; Moscou and Bogdanove 2009; Christian, Cermak et al. 2010; Li, Huang et al. 2011). Engineered TAL-nucleases are commercially available under the trademark TALEN ^™ (Cellectis, 8 rue de la Croix Jarry, 75013 Paris, France).

The rare-cutting endonuclease according to the present invention can also be a Cas9 endonuclease. Recently, novel genome engineering tools have been developed based on the RNA-guided Cas9 nuclease from the type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune system (reviewed in (Sorek, Lawrence et al. 2013)) (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012; Cong, Ran et al. 2013; Mali, Yang et al. 2013). CRISPR-associated (Cas) systems were first discovered in bacteria and used as a defense against foreign DNA (viruses or plasmids). CRISPR-mediated genome engineering initially proceeded by selecting a target sequence, typically flanked by a short sequence motif known as a protospacer adjacent motif (PAM). After target sequence selection, a specific crRNA complementary to the target sequence is engineered. The cis-activating crRNA (tracrRNA) required in CRISPR II systems pairs with the crRNA and binds to the provided Cas9 protein. Cas9 serves as a molecular anchor that facilitates base pairing between tracrRNA and crRNA (Deltcheva, Chylinski et al. 2011). In this ternary complex, the dual tracrRNA:crRNA structure serves as a guide RNA that directs the endonuclease Cas9 to the cognate target sequence. Target recognition by the Cas9-tracrRNA:crRNA complex begins by scanning the target sequence for homology between the target sequence and the crRNA. In addition to target sequence-crRNA complementarity, DNA targeting requires the presence of a short motif adjacent to the protospacer sequence (protospacer adjacent motif - PAM). After pairing between the dual-RNA and the target sequence, Cas9 then introduces a flush double-strand break three bases upstream of the PAM motif (Garneau, Dupuis et al. 2010).

Rare cutting endonucleases may be homing endonucleases, also known as meganucleases. Such homing endonucleases are well known in the art (Stoddard 2005). Homing endonucleases recognize DNA target sequences and produce single or double strand breaks. Homing endonucleases are highly specific and can recognize DNA target sites ranging from 12 to 45 base pairs (bp) in length, typically ranging from 14 to 40 bp in length. The homing endonuclease according to the present invention may correspond to, for example, LAGLIDADG endonuclease, HNH endonuclease, or GIY-YIG endonuclease. A preferred homing endonuclease according to the present invention may be an I-Crel variant.

"Delivery vehicle" refers to any delivery vehicle that can be used in the present invention to bring a cell into contact (in other words, "contact") or deliver (in other words, "introduce") the active agents/chemicals and molecules (proteins or nucleic acids) required for the present invention into a cell or subcellular compartment. This includes (but is not limited to) liposome delivery vehicles, viral delivery vehicles, drug delivery vehicles, chemical carriers, polymer carriers, lipoplexes, polyplexes, dendrimers, microbubbles (ultrasound contrast agents), nanoparticles, emulsions, or other suitable transfer vehicles. These delivery vehicles allow for the delivery of molecules, chemicals, macromolecules (genes, proteins), or other vectors such as plasmids developed by Diatos, peptides. In these cases, the delivery vehicle is a molecular vector. "Delivery vehicle" also refers to a delivery method that allows transfection.

The term "vector" refers to a nucleic acid molecule that can transport another nucleic acid to which it is linked. "Vectors" herein include, but are not limited to, viral vectors, plasmids, RNA vectors, or linear or circular DNA or RNA molecules that can be composed of chromosomal, non-chromosomal, semisynthetic, or synthetic nucleic acids. Preferred vectors are those that can replicate autonomously (episomal vectors) and/or can express nucleic acids to which they are linked (expression vectors). A large number of suitable vectors are known to those skilled in the art and are commercially available.

Viral vectors include retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza virus), baculoviruses (e.g., rabies virus and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai virus), positive-strand RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses including adenoviruses, herpesviruses (e.g., herpes simplex virus type 1 and type 2, Epstein-Barr virus, megavirus), and poxviruses (e.g., vaccinia, fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include: avian leukosis sarcoma virus, mammalian C-type virus, B-type virus, D-type virus, HTLV-BLV group, lentivirus, spumavirus (Coffin, J.M., Retroviridae: The viruses and their replication, Fundamental Virology, 3rd edition, B.N. Fields et al., eds., Lippincott-Raven Publishers, Philadelphia, 1996).

- "Lentiviral vector" means an HIV-based lentiviral vector that is extremely promising for gene delivery because of its relatively large packaging capacity, reduced immunogenicity, and its ability to stably and efficiently transduce a wide range of different cell types. Lentiviral vectors are typically produced after transient transfection of three (packaging, envelope, and transfer) or more plasmids into producer cells. Like HIV, lentiviral vectors enter target cells through the interaction of viral surface glycoproteins with receptors on the cell surface. Upon entry, viral RNA undergoes reverse transcription mediated by the viral reverse transcriptase complex. The product of reverse transcription is double-stranded linear viral DNA, which is a substrate for the integration of the virus into the DNA of infected cells. "Integrated lentiviral vector (or LV)" means, as a non-limiting example, a vector that can be integrated with the genome of the target cell. In contrast, "non-integrating lentiviral vector (or NILV)" means an effective gene delivery vector that is not integrated with the genome of the target cell through the action of the viral integrase.

- Delivery vehicles and vectors can be combined or associated with any cell penetration technology such as sonoporation or electroporation or derivatives of these technologies.

- Cell means any eukaryotic living cell, primary cell and cell line derived from these organisms for in vitro culture.

- "Primary cells" means cells taken directly from living tissue (i.e., biopsy material) and established for in vitro growth, which have undergone very few population doublings and are therefore more representative of the major functional components and characteristics of the tissue from which they are derived than persistent oncogenic or artificially immortalized cell lines.

As non-limiting examples, the cell line can be selected from the group consisting of CHO-K1 cells; HEK293 cells; Caco2 cells; U2-OS cells; NIH 3T3 cells; NSO cells; SP2 cells; CHO-S cells; DG44 cells; K-562 cells; U-937 cells; MRC5 cells; IMR90 cells; Jurkat cells; HepG2 cells; HeLa cells; HT-1080 cells; HCT-116 cells; Hu-h7 cells; Huvec cells; and Molt 4 cells.

All of these cell lines can be modified by the methods of the present invention to provide cell line models for producing, expressing, quantifying, detecting, and studying genes or proteins of interest; these models can also be used to screen for bioactive molecules of interest in a variety of fields such as, by way of non-limiting example, chemistry, biofuels, therapeutics, and agronomy, for research and production.

"Mutation" refers to a substitution, deletion, or insertion of up to one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, twenty, twenty-five, thirty, forty, fifty, or more nucleotides or amino acids in a polynucleotide (cDNA, gene) or polypeptide sequence. A mutation may affect the coding sequence of a gene or its regulatory sequences. It may also affect the structure of the genomic sequence or the structure/stability of the encoded mRNA.

- "Variant" means a repeat variant, a variant, a DNA binding variant, a TALE-nuclease variant, a polypeptide variant obtained by mutation or substitution of at least one residue in the amino acid sequence of a parent molecule.

- "Functional variant" means a catalytically active mutant of a protein or protein domain; the mutant may have the same activity or other properties as its parent protein or protein domain, or a higher or lower activity.

"Identity" refers to the sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing positions in each sequence that can be aligned for comparison purposes. When a position in the compared sequences is occupied by the same base, then the molecules are identical at that position. The degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. A variety of alignment algorithms and/or programs can be used to calculate the identity between two sequences, including FASTA or BLAST, which are available as part of the GCG sequence analysis package (University of Wisconsin, Madison, WI), and can be used, for example, with preset settings. For example, polypeptides that are at least 70%, 85%, 90%, 95%, 98% or 99% identical to a specific polypeptide described herein and preferably exhibit substantially the same function, and polynucleotides encoding such polypeptides, are contemplated.

"Similarity" describes the relationship between the amino acid sequences of two or more polypeptides. BLASTP can also be used to identify amino acid sequences that have at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, or 99% sequence similarity to a reference amino acid sequence using similarity matrices such as BLOSUM45, BLOSUM62, or BLOSUM80. Unless otherwise specified, similarity score values are based on the use of BLOSUM62. When using BLASTP, percent similarity is based on BLASTP positive scores, and percent sequence identity is based on BLASTP identity scores. BLASTP "identity" indicates the total number and fraction of identical bases in a high-scoring sequence pair; and BLASTP "positive" indicates the number and fraction of residues that are similar to each other for alignments with positive scores. Amino acid sequences having these degrees of identity or similarity, or any intermediate degrees of identity or similarity, to the amino acid sequences disclosed herein are contemplated and encompassed by the present disclosure. The polynucleotide sequences of similar polypeptides are deduced using the genetic code and can be obtained by conventional methods. Polynucleotides encoding such functional variants will be made by reverse translating their amino acid sequences using the genetic code.

- "Signal transduction domain" or "costimulatory ligand" refers to a molecule on an antigen presenting cell that specifically binds to a cognate costimulatory molecule on a T-cell, thereby providing a signal in addition to the primary signal provided by, for example, binding of the TCR/CD3 complex to a peptide-loaded MHC molecule, which mediates T cell responses including, but not limited to, proliferation, activation, differentiation, etc. Costimulatory ligands may include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecules (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, and Toll ligand receptor. Co-stimulatory ligands include, but are not limited to, antibodies that specifically bind to co-stimulatory molecules present on T cells, such as CD27, CD28, 4-IBB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

"Costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a co-stimulatory response such as, but not limited to, proliferation by the cell. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors.

As used herein, a "co-stimulatory signal" refers to a signal that, in combination with a primary signal (such as TCR/CD3 engagement), results in T cell proliferation and/or upregulation or downregulation of key molecules.

As used herein, the term "extracellular ligand-binding domain" is defined as an oligopeptide or polypeptide that can bind to a ligand. Preferably, the domain will be capable of interacting with a cell surface molecule. For example, the extracellular ligand-binding domain can be selected to recognize a ligand that acts as a cell surface marker on a target cell associated with a particular disease state. Thus, examples of cell surface markers that can act as ligands include those associated with viral, bacterial, and parasitic infections, autoimmune diseases, and cancer cells.

As used herein, the term "subject" or "patient" includes all members of the animal kingdom, including non-human primates and humans.

The above written discussion of the invention provides such means and methods for making and using the same that any person skilled in the art can make and use the same, such enablement being particularly provided for the subject matter of the appended claims forming a part of this original description.

When numerical limits or ranges are stated herein, the endpoints are included. Furthermore, all values and subranges within the numerical limits or ranges are specifically included as if expressly written.

The above discussion is presented to enable one skilled in the art to make and use the present invention, and is provided in the context of a particular application and its requirements. Various modifications to the preferred embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the present invention. Therefore, the present invention is not intended to be limited to the embodiments shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein.

Having generally described this invention, a further understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified.

Example

Example 1: Proliferation of TCRα-inactivated cells expressing 4G7-CAR.

Heterodimeric TALE-nucleases targeting two 17-bp long sequences (called half-targets) separated by a 15-bp spacer in the T-cell receptor alpha constant chain region (TRAC) gene were designed and produced. Each half-target is recognized by a repeat of the half-TALE-nuclease listed in Table 1.

Each TALE-nuclease construct was subcloned under the control of the T7 promoter in a mammalian expression vector using restriction enzyme digestion. mRNA encoding the TALE-nuclease cleaving TRAC genomic sequence was synthesized from a plasmid carrying the coding sequence downstream from the T7 promoter.

Purified T cells pre-activated with anti-CD3/CD28-coated beads during 72 hours were transfected with each of two mRNAs encoding two half TRAC_T01 TALE-nucleases. 48 hours after transfection, T cells were transduced with a lentiviral vector encoding 4G7-CAR (SEQ ID NO: 14). Two days after transduction, CD3 _NEG cells were purified using anti-CD3 magnetic beads and reactivated with soluble anti-CD28 (5 μg/ml) after the fifth day of transduction.

Cell proliferation was tracked for up to 30 days after reactivation by counting cells twice a week. Figure 1 shows the fold-induction of cell numbers relative to the number of cells present on day 2 after reactivation for two different donors. Increased proliferation was observed in non-activated cells expressing 4G7-CAR TCRα, particularly when reactivated with anti-CD28, compared to untransduced cells.

To investigate whether human T cells expressing 4G7-CAR exhibit an activated state, the expression of the activation marker CD25 was analyzed by FACS 7 days after transduction. As shown in Figure 2, purified cells transduced with a lentiviral vector encoding 4G7-CAR expressed significantly more CD25 on their surface than untransduced cells. Increased CD25 expression was observed in both CD28 reactivation and no reactivation conditions.

Example 2: Comparison of basal activation of primary human T cells expressing 4G7-CAR and classic FMC63-CAR.

To determine whether 4G7 scFV confers a prolonged "activated" state to transduced cells, basal activation of T cells transduced with CARs loaded with 4G7 scFV (SEQ ID NO: 15 encoded by SEQ ID NO: 17) or the classic FMC63 scFV (SEQ ID NO: 16) was compared.

Purified human T cells were transduced according to the following protocol: Briefly, 1×10 6 CD3+ cells preactivated with anti-CD3/CD28-coated beads and recombinant IL2 were transduced with lentiviral vectors encoding 4G7-CAR (SEQ ID NO: 15) and FMC63-CAR (SEQ ID NO: ^{16) at an MOI of 5 in 12-well non-tissue culture plates coated with 30 μg/ml recombinant retronectin over a 3-day period. 24 hours after transduction, the culture medium was removed and replaced with fresh medium. Cells were then maintained at a concentration of 1×10 6 cells} /ml by counting cells every 2 to 3 days throughout the culture period.

The percentage of cells expressing CAR was assessed by flow cytometry 3, 8, and 15 days after transduction with lentiviral vectors encoding 4G7-CAR or FMC63-CAR. It was observed that the efficiency of transduction was relatively equivalent using both lentiviral vectors (Figure 3).

We then investigated whether human T cells expressing 4G7-CAR exhibited a more activated state than human T cells expressing FMC63-CAR. To this end, we compared the expression of the activation marker CD25 on the surface of T cells transduced with the two lentiviral vectors at different time points. As shown in Figure 4, at 3 and 8 days after transduction, cells transduced with the lentiviral vector encoding 4G7-CAR expressed significantly more CD25 on their surface than cells transduced with the lentiviral vector encoding FMC63-CAR.

The size of cells transduced with 4G7-CAR or FMC63-CAR was also assessed by flow cytometry at different time points. It was observed that cells expressing 4G7-CAR were larger than cells expressing FMC63-CAR 3, 8, and 15 days after transduction (Figure 5).

After nonspecific activation in vitro, cells transduced with 4G7-CAR exhibited increased cell size (bud formation) and expression of activation markers (CD25) for a prolonged period. This long-term activation allowed for prolonged proliferation compared to cells transduced with a similar CAR containing FMC63 ScFv.

Example 3: Comparison of the proliferation of primary human T cells expressing 4G7-CAR and classic FMC63-CAR.

To determine whether 4G7 scFV confers higher proliferation activity, the proliferation of T cells transduced with CARs carrying 4G7 scFV (SEQ ID NO: 15 encoded by SEQ ID NO: 17) or classic FMC63 scFV (SEQ ID NO: 16) was tracked by counting cells twice a week for up to 20 days. Purified human T cells were transduced according to the following protocol: Briefly, lentiviral vectors encoding 4G7-CAR (SEQ ID NO: 15) and FMC63-CAR (SEQ ID NO: 16) were used to transduce 1×10 ⁶ CD3+ cells pre-activated with anti-CD3/CD28-coated beads and recombinant IL2 over a 3-day period. The cells were then maintained under typical conditions and reactivated on day 12. Cells were seeded at the same density and counted twice a week over a 20-day period. As shown in Figure 6, the proliferation activity of T cells expressing 4G7-CAR was two times higher than that of cells expressing classic FMC63-CAR.

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Claims (20)

1. A CD19-specific chimeric antigen receptor comprising at least one extracellular ligand-binding domain, a transmembrane domain, and at least one intracellular signal transduction domain, wherein the extracellular ligand-binding domain comprises a single-chain FV fragment derived from a CD19-specific monoclonal antibody 4G7, the single-chain FV fragment comprising a variable fragment of the CD19 monoclonal antibody 4G7 immunoglobulin γ1 heavy chain with an amino acid sequence as shown in SEQ ID NO:3, and a variable fragment of the CD19 monoclonal antibody 4G7 immunoglobulin κ light chain with an amino acid sequence as shown in SEQ ID NO:4 or 5. 2. The CD19-specific chimeric antigen receptor of claim 1, wherein the single-chain FV fragment comprises the amino acid sequence shown in SEQ ID NO:7 or 8. 3. The CD19-specific chimeric antigen receptor of claim 1 or 2, wherein the intracellular signal transduction domain comprises the CD3ζ signal transduction domain. 4. The CD19-specific chimeric antigen receptor of claim 3, wherein the intracellular signal transduction domain comprises a 4-1BB domain. 5. The CD19-specific chimeric antigen receptor according to claim 1 or 2, comprising a human CD8α chain transmembrane and stem domain. 6. The CD19-specific chimeric antigen receptor according to claim 1, comprising the amino acid sequence shown in SEQ ID NO: 14 or 15. 7. The CD19-specific chimeric antigen receptor according to claim 1 or 2, further comprising another non-CD19-specific extracellular ligand-binding domain. 8. A polynucleotide encoding the chimeric antigen receptor according to any one of claims 1 to 7. 9. The polynucleotide of claim 8, comprising the nucleic acid sequence shown in SEQ ID NO:17. 10. An expression vector comprising the polynucleotide of claim 8 or 9. 11. Genetically modified immune cells that express a CD19-specific chimeric antigen receptor according to any one of claims 1 to 7 on their cell surface membrane. 12. The genetically modified immune cell of claim 11, further comprising another non-CD19-specific chimeric antigen receptor. 13. The genetically modified immune cells according to claim 11, which are derived from inflammatory T-lymphocytes, cytotoxic T-lymphocytes, regulatory T-lymphocytes, or helper T-lymphocytes. 14. The genetically modified immune cell of claim 11, wherein the cell is derived from a healthy donor. 15. The genetically modified immune cell of claim 11, wherein the cell is derived from a patient diagnosed with cancer. 16. Use of the modified immune cells according to any one of claims 11 to 15 in the preparation of a medicament for treating cancer in patients in need of it. 17. The use according to claim 16, wherein the cancer is B-cell lymphoma or leukemia. 18. Methods for modifying immune cells include: (a) Provides immune cells, (b) Expressing at least one CD19-specific chimeric antigen receptor according to any one of claims 1 to 7 on the surface of the cell. 19. The method for modifying immune cells according to claim 18, comprising: (a) Provides immune cells, (b) Introducing at least one polynucleotide encoding the CD19-specific chimeric antigen receptor into the cells. (c) Express the polynucleotide into the cell. 20. The method for modifying immune cells according to claim 18, comprising: (a) Provides immune cells, (b) Introducing at least one polynucleotide encoding the CD19-specific chimeric antigen receptor into the cells. (c) Introduce at least one other chimeric antigen receptor that is not CD19 specific.